Hematopoietic stem cells (HSCs) have the extraordinary capacity to differentiate into all the cell types in the hematopoietic system while maintaining their own population through self-renewal. Disturbance in this balance between self-renewal and differentiation can lead to fatal hematologic malignancies, diseases that can be effectively treated with healthy HSCs for restoration of normal hematopoiesis and immunity. It is critical that we understand the mechanisms governing stem cell function in order to design more effective strategies to prevent their dysfunction and harness their therapeutic potential. Currently, our knowledge of marrow stem cells is based almost solely on very small subpopulations of highly purified, predominantly quiescent HSCs. However, we have shown that a significant population of cycling HSCs is lost during this purification process, and that this cycling stem cell pool can be, in part, recovered within the Lineage positive cellular fraction. Therefore, we propose that the highly purified HSCs, while exhibiting potent stem cell activity, are not fully representative of the total stem cell potential within marrow. In these studies, we propose to fully characterize a population of stem cells in marrow that are discarded with conventional HSC isolation strategies. We will determine their phenotype, cell cycle status, molecular signature and explore the mechanisms responsible for their loss during standard HSC purification strategies. We anticipate that the results of these experiments will have significant implications for the future study of HSCs. First, we are hopeful that characterization of this population of understudied stem cells within marrow will lead to a valuable expanded population of HSCs for clinical use. Second, virtually all our knowledge about HSCs--their cell cycle state, their bone marrow niche, and the transcriptional pathways governing their self-renewal and differentiation behavior--come from studies on only the highly purified HSCs. Thus, we expect that such fundamental characteristics of stem cell function will need to be re-explored within the context of the broader population of stem cells in marrow in order to gain a more comprehensive and accurate view of hematopoietic stem cell biology. My long-term career goal is to become an independent scientific investigator and leader in the field of hematopoietic stem cell biology. During my Hematology/Oncology fellowship at Brown University, and now as an Assistant Professor in the Division of Hematology/Oncology at Rhode Island Hospital, my clinical experience coupled with my research experience in Dr. Peter Quesenberry's stem cell laboratory have provided me with a strong foundation in the field of stem cell biology. This stem cell experience greatly complements my earlier graduate training in the Medical Scientist Training Program at the University of Pittsburgh, during which time I gained invaluable research experience in molecular and cellular biology. However, as a relatively new investigator in the field, I have important educational gaps that would be filled by the additional training facilitate by this career development award. Specifically, the opportunities afforded by this Mentored Career Development Award will greatly strengthen my academic, technical, statistical, and grant- writing skills, thereby greatly increasing the likelihood that I will be a successful, highl-competitive R01-funded researcher in the future.